The broad objective of this proposal is to study the regulation of the drug transporter, P-glycoprotein (PGP), by phosphorylation in multidrug- resistant (MDR) human breast cancer cells. PGP is the product of the MDR gene and is a plasma membrane-associated ATP-dependent drug efflux pump that is responsible for conferring resistance to structurally diverse natural product anticancer drugs. PGP also serves as a substrate for protein-serine kinases such as protein kinase C (PKC) and cyclic AMP- dependent protein kinase (PKA). MDR can be increased in MDR1-expressing human breast cancer cells following transfection with PKC-alpha, and this effect is associated with decreased drug retention and increased phorbol ester-stimulated PGP phosphorylation. Therefore, the hypothesis to be addressed is that PGP is modulated post-translationally by PKC and PKA in breast cancer cells. The First Aim of this proposal will be to stably transfect human MCF-7 breast carcinoma cells which over-express PGP but contain low PKC-alpha and type I PKA activities (BC-19 cells) with wild type or constitutively active mutant forms of PKC-alpha, the catalytic fragment of PKC-alpha (PKM) or the catalytic subunit of PKA, to determine their effects on the MDR phenotype. In instances where a particular PKC isoform is less abundant in MDR breast cancer cells (MCF-7/ADR cells), eg. PKC-epsilon, MDR cells will be transfected with the low abundance form of PKC and its effect on the MDR phenotype determined. The Second Aim will be to inhibit selectively PKCalpha, which is over-expressed in MCF-7/ADR cells, by stably expressing antisense PKCalpha cDNA constructs. New PKC inhibitor analogs will also be assessed for their selectivity in inhibiting PKC isoforms and for their ability to reverse MDR in MCF-7/ADR and BC-19 cells. The role of elevated type PKA in MCF-7/ADR cells will also be determined by selectively down-regulating its activity with 8-C1- cAMP, which binds selectively to the type I cAMP-binding subunit of PKA (R-I), or with an antisense oligodeoxynucleotide (OGN) against R-I. The Third Aim will be to determine the effect of mutating the known or consensus PKC and PKA consensus phosphorylation sites in PGP on its ability to confer MDR to wild type or PKC-alpha-overexpressing MCF-7 cells.